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. 2001 Sep 17;20(18):5165–5175. doi: 10.1093/emboj/20.18.5165

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Copyright © 2001 European Molecular Biology Organization

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graphic file with name cde497f1a.jpg — Fig. 1. Interaction of Hct1 with Clb2 and the APC. Whole-cell extracts (WCE) and proteins immunoprecipitated with antibodies to HA (α-HA IP) or Myc (α-Myc IP) were analysed by immunoblotting with HA- (α-HA), Myc- (α-Μyc) or Cdk1- (α-Cdk1) specific antibodies. (A) Hct1 binds Clb2–Cdk1 complexes. cdc23-1 strains carrying CLB2 or CLB2-myc12 and expressing untagged (–) or HA3-tagged (+) HCT1 from the GAL1 promoter were grown to exponential phase at 25°C and treated with galactose for 2 h. Lane 1, W989; lane 2, W987; lane 3, W1067; lane 4, W1066. (B) Phospho-Hct1 binds Clb2 but not the APC. Wild-type (no myc tag), CDC23-myc9 and CLB2-myc12 strains lacking (–) or containing (+) GALL-HA3-HCT1 were grown to exponential phase (C) or arrested with nocodazole for 3 h (N) and then treated with galactose for 2 h. Lanes 1 and 2, K699, lanes 3 and 4, W1158, lanes 5 and 6, W1641, lanes 7 and 8, W1644, lanes 9 and 10, W1079, lanes 11 and 12, W1156. (C) Hct1 binds Clb2 at normal protein levels. Wild-type strains carrying CLB2 or CLB2-HA3 and untagged (–) or Myc18-tagged (+) HCT1 were arrested with nocodazole for 3 h. Lane 1, K699; lane 2, W2541; lane 3, W9317; lane 4, W2542.

graphic file with name cde497f1b.jpg — Fig. 1. Interaction of Hct1 with Clb2 and the APC. Whole-cell extracts (WCE) and proteins immunoprecipitated with antibodies to HA (α-HA IP) or Myc (α-Myc IP) were analysed by immunoblotting with HA- (α-HA), Myc- (α-Μyc) or Cdk1- (α-Cdk1) specific antibodies. (A) Hct1 binds Clb2–Cdk1 complexes. cdc23-1 strains carrying CLB2 or CLB2-myc12 and expressing untagged (–) or HA3-tagged (+) HCT1 from the GAL1 promoter were grown to exponential phase at 25°C and treated with galactose for 2 h. Lane 1, W989; lane 2, W987; lane 3, W1067; lane 4, W1066. (B) Phospho-Hct1 binds Clb2 but not the APC. Wild-type (no myc tag), CDC23-myc9 and CLB2-myc12 strains lacking (–) or containing (+) GALL-HA3-HCT1 were grown to exponential phase (C) or arrested with nocodazole for 3 h (N) and then treated with galactose for 2 h. Lanes 1 and 2, K699, lanes 3 and 4, W1158, lanes 5 and 6, W1641, lanes 7 and 8, W1644, lanes 9 and 10, W1079, lanes 11 and 12, W1156. (C) Hct1 binds Clb2 at normal protein levels. Wild-type strains carrying CLB2 or CLB2-HA3 and untagged (–) or Myc18-tagged (+) HCT1 were arrested with nocodazole for 3 h. Lane 1, K699; lane 2, W2541; lane 3, W9317; lane 4, W2542.